1. Field of the Invention
This invention relates to a method, device and kit for estimation of total protein in a sample.
2. Description of the Related Art
Proteins are the essential components of life processes and thus play a central role in biological research as well as many commercial processes. The study and use of proteins inevitably requires quantifying the amount of protein in a sample.
Over the last hundred years several methods have been developed for estimation of total protein concentration, also referred to herein as protein assay. The biuret, refractometric and total nitrogen methods are amongst the earliest examples of protein estimation. The biuret method relies on the reaction of alkaline copper with the peptide bonds of protein which gives a characteristic purple color; unfortunately this method is not very sensitive for total protein estimation and thus it is not widely used.
At the turn of this century, Folin and Denis (J. Biol. Chem., 12:239-243 (1912), incorporated herein by reference) developed a phospho-molybdictungstic acid, commonly known as Folin reagent, which shows a strong color reaction with many phenol derivatives, including tyrosine and tryptophan. The development of the Folin reagent began an intense era in the development of coloriinetric protein assays using the Folin reagent, as demonstrated by the publication of such methods by a number of researchers, including Greenberg (J. Biol. Chem., 82:545 (1929), and Folin and Ciocalteu (J. Biol. Chem., 73:627 (1927)), each of which is incorporated herein by reference. Then, in 1951, Lowry et al (J. Biol. Chem. 193:265-275, incorporated herein by reference) published their classical and comprehensive work for protein estimation using the Folin reagent, known as the Lowry method. From 1951 until the present, the Lowry method, including various modifications thereof, remained unrivaled and is currently regarded as the standard method for protein estimation.
Despite the wide application of the Lowry method over the last fifty years, the Lowry method suffers from numerous and well recognized drawbacks. In a nutshell, the Lowry method simply does not work well in most situations, i.e. the method is unreliable and suffers from interference with numerous commonly used laboratory agents (see, e.g., Peterson, G. L, Anal.
Biochem. 100:201-220 (1979), Peterson, G. L., Methods in Enzymology 91:95-119 (1983), and Stoscheck, C. M., Methods in Enzymology, 182:50-68 (1990), each of which is incorporated herein by reference). The difficulties in overcoming the drawbacks of the Lowry method are evidenced by the fact that despite a period of staggering growth in the biological sciences, the art of protein estimation remains limited to the Lowry method.
A. Long Sought And Unresolved Needs and Failures of Others
In 1979, Gary L. Peterson (Anal. Biochem. 100:201-220) published a review comparing the Lowry method with other protein assays. The review, which is herein incorporated by reference, included cross references to 127 publications describing the work of several hundred researchers. In his review, Peterson stated that "the principle disadvantage of this [Lowry] method is its lack of specificity." Peterson listed several hundred compounds and their derivatives that interfere with the Lowry method. Peterson also discussed numerous modifications of the Lowry method, including one of his own work, that he had tried; these methods achieved only limited success at best in improving the specificity and reducing interference by laboratory reagents.
The Peterson reference also provides an overview, as of 1979, of all other reported methods for protein determination, including biuret, Kjeldahl, U.V. absorption, alkaline hydrolysis-ninhydrin, protein fluorescence, turbidimetric, and protein-dye binding methods. All these methods suffer from a lack of specificity, i.e., large protein-to-protein variation, as well as poor sensitivity and complicated protocols. A relatively popular dye binding method by Bradford (Anal Biochem. 72:248-254 (1976), incorporated herein by reference) exhibits large protein to protein variation and interference with commonly used detergents. Other problems with the Bradford method are: (1) that the color produced is not stable due to acid precipitation of protein; and (2) that some proteins simply do not produce a linear standard curve, resulting in serious error. Peterson concluded that "all other methods seriously lack one or more of the following attributes: simplicity, sensitivity, or precision. All methods lack complete specificity (i.e., show large protein-to-protein variation and interference with common laboratory agents)."
After the publication of Peterson's review on the art of protein assay in 1979, those skilled in the art of protein assay continued trying to develop a simple, sensitive and highly specific protein assay, i.e., a protein assay independent of protein to protein variation and substantially free from interference by common laboratory agents. For example, various groups developed protein assays which involve spotting aliquots of protein solutions on support membranes.
Illustrative of this approach are articles by Kuno and Kihara (Nature 215: 975-976 (1967)), Bramhall et al (Anal. Biochem. 31:146-148 (1969)), and Minamide and Bamburg (Anal. Biochem. 190:66-70 (1990)), each of which are incorporated herein by reference. These articles describe methods of protein assay in which protein solution is spotted on filter paper. The resulting protein spots are stained with a protein dye, the dye, bound to the protein, is eluted from the filter paper, and finally the eluted dye is calorimetrically measured. All these methods are essentially variations of colorimetric methods and require the use of a standard protein and a calorimeter. In addition, they suffer from protein-to-protein variation because protein and dye interaction is not quantitative.
Kumar et. al (Biochem. Biophys. Res. Comm. 131:883-891(1985), incorporated herein by reference), have also described a protein assay in which protein is spotted on nitrocellulose paper, the resulting protein spots are stained, and protein concentration is then determined by measuring the color intensity of the protein spots. The Kumar method suffers from several drawbacks, including: complicated procedures, use of expensive instruments (e.g., a densitometer) and standards, lack of specificity, poor sensitivity, and protein-to-protein variation since the intensity of color of the protein spots depends on the tyrosine content of the protein(s) in the sample and the formation of a complex with starch. Furthermore, small protein fragments lacking tyrosine can not be assayed with this method.
These and other efforts were unsuccessful in developing a protein assay independent of protein to protein variation and substantially free from interference by common laboratory agents as demonstrated by reviews of the state of the art in 1983 and 1990 by Peterson and Stoscheck, respectively. In 1983, Peterson concluded that "unfortunately all [total protein assay] procedures that are relatively simple to perform and are usable in a variety of experimental situations do not give the same response with different proteins" (Peterson, G. L., Methods in Enzymology 91:95-119 (1983) (emphasis added), incorporated herein by reference). Similarly, in 1990, Stoscheck reviewed the sensitivity of various protein assays to common laboratory agents and the extent of protein to protein variation in these assays. (Stoscheck, C. M., Methods in Enzymology, 182:50-68 (1990), incorporated herein by reference.)
Applicant has also surveyed hundreds of papers on protein assay published until the filing of the parent patent application in 1995. Most of these articles are cited in the review articles already cited in this application. Most of these papers are either aimed at a specialized use of protein assay having limited application or describe attempts to improve existing methods. Unfortunately, they have all failed to achieve the attributes listed by Peterson and long sought by the artisan of protein assay.
In conclusion, the cited reviews establish how a large number of protein scientists over the years tried and failed to develop a reliable protein assay that is substantially protein specific, shows little or no protein-to-protein variation, is substantially free from interference by commonly used laboratory agents, is simple to perform, highly sensitive, and is easy to modify. Thus, there is a need for a protein assay with these attributes and which additionally does not require complicated procedures, running a set of known standards, or the use of colorimeters or densitometer. It is also desirable that such an assay be easily modified to allow its use in a variety of applications.
B. Historical landmark
The parent application (U.S. Ser. No. 08/370,685) of the instant application discloses a landmark development in protein chemistry, i.e., a protein assay that is substantially specific to protein with little or no protein-to-protein variation, substantially tolerates a wide variety of commonly used laboratory agents, is simple to perform, requires minimal use of instrumentation, sensitivity down to nanograms and produces substantially reliable results. As discussed above, such an assay has been sought by protein chemists for well over a century, during which thousands of scientists tried and failed to develop an assay having the above attributes. The novel protein assay described in U.S. Ser. No. 08/370,685 involves applying a small aliquot of a protein solution to a test strip or support membrane to produce a compact protein spot of a size that is substantially proportional to the concentration of protein in the protein solution. This proportional relationship allows the concentration of protein in a sample to be determined.